Progress Reports

Reporting Period:

Year 1

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Adoptive immunotherapy with functional T cells is a potentially effective therapeutic strategy against various types of cancers and viral infections. A major challenge however lies with the “exhaustion” (loss of cytotoxic and proliferative capacities) of antigen-specific T cells during expansion in culture. For an effective adoptive immunotherapy, what we need is not the "exhausted" T cells, but large number of "young and active" CD8+ T cells that can kill tumors or virus infected cells efficiently. To address this issue, we generated induced pluripotent stem cells (iPSCs) from EBV-specific CD8+ T cells from an EBV-infected patient. We then redifferentiated these iPSCs into CD8+ T cells or we like to call them “rejuvenated” T cells since they are newly generated and highly proleferative. These rejT cells possessed antigen-specific killing activity and exhibited TCR gene rearrangement patterns identical to those of the original T cell clone from the patient. In order to confirm in vivo efficacy of these rejT cell, we innoculated EBV-induced tumors into immunodeficient mice and after confimation of tumor growth, we injected these rejT cells. Results indicated that these rejT cells eliminated tumors more efficiently than the original EBV-specific CD8+ T cells, thus confirming in vivo efficacy of these T cells.
Another aspect we worked on is generation of a functional organ in livestock animals. In the past, we have demonstrated generation of rat pancreas in mouse utilising a method called "blastocyst complementation". In ancillary work, we successfully generated exogenous-pig pancreata using the same principle. Whilst these studies prepared us to examine the feasibility of generating human PSC-derived pancreata in pancreatogenesis-disabled pigs, some ethical issues on making such “admix chimeras” have yet to be solved. A part of the concern comes from the possibility that human iPSC-derived cells contribute to neural or germ cells in chimeric animals. To overcome this issue, we attempted to restrict differentiation of PSC-derived cells into endodermal organs by introducing a gene that is important for the development of internal organs. When the expression of this gene was induced after transfer of embryo to foster mother, differentiation of ES-derived cells were directed toward interenal organs avoiding contribution of those cells in germ cells, skin and nervous systems. We termed this type of organ generation as "Targeted organ generation" and this should, in principle, reduce the ethical concern when making human-livestock chimeras.
In addition, we found that the inhibition of nuclear translocation of a molecule called b-CATENIN enhances conversion of mouse EpiSCs (non-chimera forming) to naive-like PSCs (chimera forming). Since most human ES/iPSCs are considered EpiSCs and non-chimera forming, the finding is of importance for the generation of human organs in ivestock animals.

Reporting Period:

Year 2

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In this project, utilizing stem cells, we are trying to generate not only cells for cell therapy but transplantable organs for those with end stage organ failure. The following is a summary of our progress made in the generation of cells and organs from stem cells.
Adoptive T-cell immunotherapy is a potential therapeutic strategy for combating various types of cancer. However, highly expanded T cells have not proven particularly effective. This is in part explained by exhaustion and losses of function that occur during the ex vivo expansion of T cells. For an effective adoptive immunotherapy, what we need is not the "exhausted" T cells, but large numbers of "young and active" T cells that can kill tumors. To address this issue, we have recently developed a novel system in which antigen-specific killer T cells (CTLs) can be rejuvenated by reprogramming them to induced pluripotent stem cells (iPSCs) and redifferentiating them while expanding their numbers, yielding abundant rejuvenated T cells (rejT cells). We confirmed the in vivo efficacy of these rejT cell against EBV-induced tumors inoculated in immunedeficient mice. To increase the safety of this novel rejT cell therapy, we inserted a drug inducible suicide system to T cell-derived iPSCs. Both the iPSCs and rejT cells derived from them induced cell death after administration of the inducing drug in vivo thereby demonstarting efficacy of this safety system. The results facilitate clinical application of the rejT cell therapy and other branches of iPSC-derived regenerative medicine.
To help patients with end-stage organ failure waiting for organ transplantation, we are trying to generate transplantable organs in livestock animals. Previously we have demonstrated the generation of rat pancreas in mouse using interspecific blastocyst complementation technique. This time, we succeeded in the generation of mouse pancreas in rats. The generated pancreata were composed of mouse PSC-derived cells but were of rat size. Islets were prepared from these pancreata and transplanted into diabetic mice. Transplanted islets successfully normalized and maintained host blood glucose levels for over 370 days. Because the transplanted islets contained small number of rat cells, immunosuppressive drug was given to the recipient mice for the first 5 days, but the rest of time, it was totally immunosuppression free. These data provide proof-of-principle evidence for the therapeutic potential of PSC-derived islets generated in a xenogeneic host using the blastocyst complementation technique, our ultimate goal.
In order to apply this principle to generation of human organs, human PSCs that can contribute to chimera formation are necessary. However, recent studies demonstrated that currently available human PSCs are epiblast-stage stem cells (EpiSCs) and not able to contribute to chimera formation. Using a rodent model, we discovered that if cell survival is transiently promoted by over expressing the anti-apoptotic gene (BCL2), EpiSCs and even lineage-committed progenitors can contribute to chimera formation upon injection into preimplantation embryos. We similarly showed that over expressing BCL2 in human PSCs could increase their survival following injection into sheep embryos in vitro. Having successfully engineered a platform to delete the pancreas in sheep using CRISPRs, we are now in a position to evaluate how well these modified human PSCs can contribute to pancreas formation in vivo. We believe application of our newly designed strategies may generate human PSC-derived organs whilst avoiding unwanted contributions to host gametes or neural tissues.

Grant Application Details

Application Title:

Generation of functional cells and organs from iPSCs

Public Abstract:

The development of induced pluripotent stem cell (iPSC) technology may be the most important advance in stem cell biology for the future of medicine. This technology allows one to generate a patient’s own pluripotent stem cells (PSCs) from skin or blood cells. iPSCs can then be reprogrammed to multiply and produce high quality mature cells for cell therapy. Because iPSCs
are derived from a patient's own cells, therapies that use them will not stimulate unwanted immune reactions or necessitate lifelong immunosuppression. If organs can be generated from iPSCs, many patients with organ failure awaiting transplants will be helped. The goal of this project is to further develop iPSC technology to bring about personalized regenerative medicine for treating intractable diseases such as cancers, viral infections, genetic blood disorders, and organ failure. Specifically, we would like to establish three major core programs for generating from iPSCs: personalized immune cells; an unlimited supply of blood stem
cells; and functional organs.

First, we will generate iPSC-derived immune cells that kill viruses and cancer cells. Current immunotherapy uses immune cells that are exhausted (have limited ability to function and proliferate) after they multiply in a test tube. To supply active nonexhausted immune cells, iPSCs will be generated from a patient’s immune cells that target tumor cells and infections and then redifferentiated to mature immune cells with the same targets.

Second, we aim to develop iPSC technology to generate blood stem cells that replenish all blood cells throughout life. Harvesting blood stem cells from a leukemia patient for transplantation back to the patient after chemotherapy and radiation has been challenging because few blood stem cells can be harvested and may be contaminated with cancer cells. Alternatively, transplanting blood stem cells from cord blood or another person requires genetic matching to prevent immune reactions. However, generating blood stem cells from a patient’s iPSCs may avoid contamination with cancer cells, immune reactions, and
the need to find a matched donor. Furthermore, we aim to generate iPSCs from a patient with a genetic blood disease, correct the genetic defect in the iPSCs, and generate from these corrected iPSCs healthy blood stem cells that may be curative when transplanted back into the patient.

Lastly, we will try to generate from iPSCs not just mature cells, but organs for transplantation, to potentially address the tremendous shortage of donated organs. In a preliminary study, we generated preclinical models that could not develop pancreases. When we injected stem cells into these models, they developed functional pancreases derived from the injected cells and survived to adulthood. We hope that within 10 years, we will be able to provide a needed organ to a patient by growing it from the patient’s own PSCs in a compatible animal.

Statement of Benefit to California:

Cancer is the second leading cause of death, accounting for 24% of all deaths in the U.S. Nearly 55,000 people will die of the disease--about 150 people each day or one of every four deaths in California. In 2012, nearly 144,800 Californians will be diagnosed with cancer. We need effective treatment to cure cancer.

End-stage organ failure is another difficult disease to treat. Transplantation of kidneys, liver, heart, lungs, pancreas, and small intestine has become an accepted treatment for organ failure. In California, more than 21,000 people are on the waiting lists at transplant centers. However, one in three of these people will die waiting for transplants because of the shortage of donated
organs. While end-stage renal failure patients can survive for decades with hemodialysis treatment, they suffer from high morbidity and mortality. In addition, the high medical costs for increasing numbers of dialysis patients is a social issue. We need to find a way to increase organs that can be used for transplantation. In our proposed projects, we aim to use iPSC technology and recent discoveries to develop new methods for treating cancers,
viral infections, and organ failure. More specifically, we will pursue our recent discoveries using iPSCs to: (1) multiply person’s T cells that specifically target cancers and viral infections; (2) generate normal blood-forming stem cells that can be transplanted back into a patient to correct a blood disease (3) regenerate tissues and organs from a patient’s cells for transplantation back into that patient.

These projects are likely to benefit the state of California in several ways. Many of the methods, cells, and reagents generated by this research will be patentable, forming an intellectual property portfolio shared by the state and the institutions where the research is performed. The funds generated from the licensing of these technologies will provide revenue for the state, will help
increase hiring of faculty and staff (many of whom will bring in other, out-of-state funds to support their research), and could be used to ameliorate the costs of clinical trials--the final step in translation of basic science research to clinical use. Most importantly, this research will set the platform for stem cell-based therapies. Because tissue stem cells are capable of lifelong
self-renewal, these therapies have the potential to provide a single, curative treatment. Such therapies will address chronic diseases that have no cure and cause considerable disability, leading to substantial medical expenses and loss of work. We expect that California hospitals and health care entities will be first in line for trials and therapies. Thus, California will benefit economically and the project will help advance novel medical care.